Method and composition for clonal propagation of pandanus amaryllifolius

ABSTRACT

The present invention provides a method of clonal propagation of  Pandanus amaryllifolius  and further provides a composition for the clonal propagation of scented  Pandanus amaryllifolius.

FIELD OF THE INVENTION

The present invention relates to a method of clonal propagation ofPandanus amaryllifolius. The present invention relates to a compositionfor the clonal propagation of scented Pandanus amaryllifolius.

BACKGROUND ART

Pandanus amaryllifolius is a member of the family Pandanaceae comprisinga group of plants generally known as ‘screw pines’. Earlier studies byButtery et al (Chem. Ind. London, Vol. 23, page 478 of year 1982)indicated that 2-acetyl-1-pyrroline is the major flavour constituent ofPandanus leaves, while other constituents like volatile oils, alcohols,aromatic aldehydes, ketones and esters were also traceable. The majorcompound, 2-acetyl-1-pyrroline, has commercial significance as a majorflavour constituent that impart the characteristic aroma of scented ricevarieties such as Basmati rice of India, ‘Khao Dawk Mali-105′ ofThailand and Kaorimai of Japan. The essential oil of Pandanus is knownto impart 10-times more flavour than the highly scented rice and100-times more than that present in normal rice (Chem. Ind. London, Vol.23, page 478 of year 1982). Pandanus extract has been reported topossess anti-oxidant properties(http://www-ang.kfunigraz.ac.atengl/Pand_ama.html). Pandanus leaf iscited as an ingredient in many Indian, Thai and Indonesian recipes.Pandanus amaryllifolius is not known to grow in wild state whereas theplants are grown as ornamentals in pots or grown in kitchen gardens inparts of South East Asia, Indonesia and New Guinea. The plant producesonly 6-8 suckers per year and is known to produce male flowers only inNew Guinea. Female flowers are not known in the variety that synthesizesaroma in the leaves (http://www-ang.kfunigraz.ac.atengl/Pand_ama.html).Thus the lack of its wild population or the large-scale cultivation onone hand and a high potential for its economic importance on the otherhand indicate a huge future demand for this plant material. Thus therecould be possibilities of its organized cultivation and hence would be ademand for the planting material. To meet such demands, a method forrapid production of plantlets by tissue culture means has tremendousscope. Since the flavor compound in this plant is known to vary in eachvariety (http://www-ang.kfunigraz.ac.atengl/Pand_ama.html), the plantmaterial with highest perceivable flavor level was selected and a methodfor rapid multiplication of plantlets was invented. Plant Tissue Culture(PTC) is a major area of plant biotechnology, which has direct impact onpresent day agriculture/horticulture. The need for enormous supply ofdesired type of planting/sowing material may be obtained by applyingtissue culture method as an alternative to conventional method. Atpresent, several laboratories in the world are producing over 650million plants annually, by applying tissue culture method.

There are various advantages of tissue culture (TC) plants over thoseproduced by conventional methods of plant propagation. As TC plants areproduced in completely controlled environment, the chances of carryingsystemic diseases are rare. The plants are induced to multiply at atremendous rate by developing specific medium formulation for each plantmaterial. Maximum number of plants can be produced using minimum space,time and nutrients. Similarly, storage and transportation can be handledwith lesser cost than for conventional plants. It facilitates theavailability of planting material throughout the year.

The present invention relates to a medium formulation for the clonalpropagation of scented Pandanus amaryllifolius Roxb. wherein thedevelopment of specific nutrient medium with growth regulators helpestablishment of shoot cultures and rapid further aseptic multiplicationof Pandanus shoots which may subsequently be hardened to obtain highquality Pandanus planting material.

There are no reports on the tissue culture studies or clonal propagationof Pandanus amaryllifolius. However there are certain studies on thechemical composition of the plant at different stages of its life cycle.

Laksanamai V. and Ilangantilake S. (Cereal Chemistry Vol. 70 (4) 1983)found 2-acetyl-1-pyrroline in Pandanus leaves at levels 1 ppm (on freshweight basis) and described this aroma as that present in scented ricevarieties.

Another flavour, ethyl formamide present in rice as a flavouringcompound has been identified in Pandanus leaves (Naturwissenchaften,Vol. 71: 215, 1984). Yet another study found 3-methyl-2-(5H)-furanone asthe main volatile compound in Pandanus leaves, besides 3-hexanol,4-methylpentanol, 3-hexanone and 2-hexanone (Flavour Chemistry of ethnicfoods—Proceedings of a Meeting held during the 5^(th) Chemical Congressof North America, Cancun, Nov. 11-15, 1997 (1999)).

The drawback associated with the methods followed hitherto for thesupply of planting material is that of the conventional type ofpropagation involves injury to the mother plant for collecting thelateral shoot suckers which is often associated with the sacrificing ofthe selected high yielding mother plant. Conventional method ofpropagation transmits systemic infection present in the mother plantacquired from one generation to the next leading to diseased plantingmaterial. The rate of multiplication is slow.

OBJECTS OF THE PRESENT INVENTION

The main object of the present invention is to provide a method for themicro-propagation of scented Pandanus amaryllifolius through tissueculture.

An object of the present invention is to provide a method for theestablishment of aseptic shoot cultures of Pandanus using both terminaland lateral shoot buds as explants and using tissue culture methods.

Yet another object of the present invention is to provide a method touse suitable tissue culture medium for the micro propagation of scentedPandanus amaryllifolius under controlled propagation conditions.

Still yet another object of the present invention is to provide methodfor Pandanus plantlet production for the exchange and conservation ofdisease-free Pandanus germplasm.

It is also an object of the present invention is to provide acomposition congenial for the micro-propagation of Pandanus, whichobviates the drawbacks as detailed above.

Further object of the present invention is to provide a nutrientcomposition for the establishment of aseptic shoot cultures of Pandanususing both terminal and lateral shoot buds as explants and using tissueculture methods.

Yet another object of the present invention is to provide the mostsuitable composition as well as growth regulators composition formaximum multiplication of shoot cultures. Still yet another object ofthe present invention is to provide the best combination of light andtemperature conditions for maximizing the multiplication of the shootcultures using the proposed culture medium.

Further object of the present invention is to provide the nutrientrequirements for the development of roots from micro-propagated shootcultures to facilitate easy establishment of Pandanus plantlets whentransferred to soil.

SUMMARY OF THE INVENTION

The present invention also relates to a method of clonal propagation ofPandanus amaryllifolius. The present invention also provides acomposition for the clonal propagation of scented Pandanusamaryllifolius.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, the present invention provides a method for themicro-propagation of scented Pandanus amaryllifolius through tissueculture, said method comprising the steps of:

-   -   (a) cutting one or more plant explants of Pandanus        amarylifolius;    -   (b) removing any soil and other contaminants from the surface of        the explants;    -   (c) cutting the decontaminated explants in laminar flow;    -   (d) culturing the explants for three-eight weeks is at 20-35        degree C. in dark in a medium consisting of following        ingredients NH₄NO₃, in the range of 750-2000; KNO₃, 800-1800;        H₃BO₄, 6.2; KH₂PO₄, 170.0; KI, 0.83; Na₂MoO₄, 0.25; CoCl₂,        0.025; CaCl₂ 2H₂O, 440-800; MgSO₄ 5H2O, 0.025; Na₂ EDTA, 37.35;        FeSO₄ 7H₂O, 27.85; Thiamine HCl, 1.00; Pyridoxine HCl, 1.00;        Nicotinic acid, 1.00; Glycine, 4.00; Inositol, 100.00; Ascorbic        acid, 50.00-250.00; Glutamine, 50-200; Sucrose, 10000-40000;        Soluble polyvinyl pyrrolidone 500-4000; Benzyl amino purine,        0.01-2.0; GELRITE-™ (phytagel), 2000.00, added in weight as        milligrams and the final volume made up to one litre by adding        glass distilled water at a pH in the range of 5.4 to 6.2 and        sterilized by autoclaving, to obtain organized shoot bud/shoot,    -   (e) continuing the culture of the explants until organized shoot        buds/shoots are formed;    -   (f) harvesting the shoot buds/shoots thus formed;    -   (g) culturing the shoots as (f) in a second medium consisting of        following ingredients NH₄NO₃, in the range of 1000-2500,        KNO_(3, 500)-2000; H₃BO₄, 6.2; KH₂PO₄, 170.0; KI, 0.83; Na₂MoO₄,        0.25; CoCl₂, 0.025; CaCl₂ 2H₂O, 440.00; MgSO₄ 7H₂O, 370.00;        MnSO₄ 4H₂O, 22.30; MnSO₄ 4 H₂O, 22.30; ZnSO₄, 8.60; CuSO₄ 5H₂O,        0.025; Na₂ EDTA, 37.35; FeSO₄ 7H₂O, 27.85; Thiamine HCl, 1.00;        Pyridoxine HCl, 1.00; Nicotinic acid, 1.00; Glycine, 4.00;        Inositol, 100.00; Ascorbic acid, 50.00-250.00; Sucrose,        10000-30000.00; Kinetin, 0.02-4.0; Benzyl amino purine, 0.5-5.0;        Agar, 7000.00-12000, added in weight as milligrams and the final        volume made up to one litre by adding glass distilled water:at        20-35 degree C. in the presence of cool white light of about        1500-6000 lux of 16 hour photoperiod for about three to six        weeks, at a pH in the range of 5.4 to 6.2 and sterilized by        autoclaving to induce multiple shoots;    -   (h) transferring the small clusters back to the second medium to        propagate shoots;    -   (i) transferring the elongated shoots to a third medium        comprising about 50% of the salts of the second medium without        growth regulator and 30-80% sucrose of the second medium, all        dissolved in tap water, at a pH in the range of 5.4 to 6.2,        10000 mg per liter of agar and sterilized by autoclaving; and    -   (j) culturing the shoots of step (i) at about 20-35 degree C. in        the presence of cool white light of about 1500-6000 lux of 16        hour photoperiod for at least three weeks, to induce roots and        to partially harden the individual shoots and further to obtain        a large number of partially hardened shoots by tissue culture        for transferring to the soil.

Yet another embodiment of the present invention, wherein the plantexplants are selected form axillary/terminal shoot tips/buds of Pandanusamaryllifolius plants.

Further embodiment of the present invention, wherein at least one plantgrowth regulator that is employed in the medium of step (d) and step (g)is selected from the group consisting of auxins and cytokinins, and acombination thereof.

Still another embodiment of the present invention, wherein the growthregulator auxin is selected from the group consisting of indole aceticacid, indole butyric acid and naphthalene acetic acid, at aconcentration range varying between 0.01 to 10 mg/L w/v. It is also anembodiment of the present invention, wherein the growth regulatorcytokinins is selected from the group consisting of6-benzylamino-purine, gamma-gamma-dimethyl allylamino purine,isopentinyl adenine and kinetin, at a concentration range varyingbetween 0.01 mg/L up to 20 mg/L.

Yet another embodiment of the present invention, wherein thedecontamination of the small tissue is achieved by dipping in a solutioncontaining at least one sterilizing agent selected from the groupconsisting of sodium hypochlorite, calcium hypochlorite, mercuricchloride, and ethyl alcohol.

Yet another embodiment of the present invention, wherein said methodfacilitates shoot multiplication to the extent of 10 fold in about 4weeks time period.

Still another embodiment of the present invention, wherein said methodfacilitates production of Pandanus amaryllifolius plantlets free fromsaprophytic, fungal and bacterial diseases.

Further embodiment of the present invention, wherein explants fromPandanus shoot axis are surface sterilized, aseptically dissected inlaminar air-flow and meristems/shoot buds are isolated for transfer toMurashige and Skoog's tissue culture medium with 3% w/v of sucrose asthe carbon source and various combinations of growth regulators forshoot bud establishment.

In another embodiment of the present invention the cultured shoot budsare subjected for shoot multiplication using Murashige and Skoog'snutrient medium with changes in the concentrations of growth regulatorsto induce multiple shoot cultures.

In yet another embodiment of the present invention, the multiple shootsmay aseptically be transferred to yet another Murashige and Skoog'smedium with reduced levels of nutrients to induce roots helpful forsubsequent transfer of the plantlets to soil.

Yet another embodiment of the present invention, wherein thedecontamination of the small tissue is achieved by dipping in a solutioncontaining at least one sterilizing agent selected from the groupconsisting of sodium hypochlorite, calcium hypochlorite, mercuricchloride, and ethyl alcohol.

In an embodiment of the present invention explants from Pandanus shootaxis are surface sterilized, aseptically dissected in laminar air-flowand meristems/shoot buds are isolated for transfer to Murashige andSkoog's tissue culture medium with 3% w/v of sucrose as the carbonsource and various combinations of growth regulators for shoot budestablishment.

In another embodiment of the present invention the cultured shoot budsare subjected for shoot multiplication using Murashige and Skoog'snutrient medium with changes in the concentrations of growth regulatorsto induce multiple shoot cultures.

In yet another embodiment of the present invention, the multiple shootsmay aseptically be transferred to yet another Murashige and Skoog'smedium with reduced levels of nutrients to induce roots helpful forsubsequent transfer of the plantlets to soil.

The present invention also provides composition for inducing multipleshoots for the micro-propagation of scented Pandanus amaryllifolius,said composition comprising: NH₄NO₃, in the range of 750-2000,KNO_(3, 800)-1800; H₃BO₄, 6.2; KH₂PO₄, 170.0; KI, 0.83; Na₂MoO₄, 0.25;CoCl₂, 0.025; CaCl₂ 2H₂O, 440-800; MgSO₄ 7H₂O, 370.00; MnSO₄ 4H₂O,22.30; MnSO₄ 4 H₂O, 22.30; ZnSO₄, 8.60; CuSO₄ 5H₂O, 0.025; Na₂ EDTA,37.35; FeSO₄ 7H₂O, 27.85; Thiamine HCl, 1.00; Pyridoxine HCl, 1.00;Nicotinic acid, 1.00; Glycine, 4.00; Inositol, 100.00; Ascorbic acid,50.00-250.00; Glutamine, 50-200; Sucrose, 10000-40000; Soluble polyvinylpyrrolidone 500-4000; Benzyl amino purine, 0.01-2.0; GELRITE-™(phytagel), 2000.00 all added in weight as milligrams and the finalvolume made up to one litre by adding glass distilled water. Anembodiment of the present invention, a composition for inducing multipleshoots for the micro-propagation of scented Pandanus amaryllifolius,said composition comprising: NH₄NO₃, in the range of 1000-2500, KNO₃,500-2000; H₃BO₄, 6.2; KH₂PO₄, 170.0; KI, 0.83; Na₂MoO₄, 0.25; CoCl₂,0.025; CaCl₂ 2H₂O, 440.00; MgSO₄ 7H₂O, 370.00; MnSO₄ 4H₂O, 22.30; MnSO₄4 H₂O, 22.30; ZnSO₄, 8.60; CuSO₄ 5H₂O, 0.025; Na₂ EDTA, 37.35; FeSO₄7H₂O, 27.85; Thiamine HCl, 1.00; Pyridoxine HCl, 1.00; Nicotinic acid,1.00; Glycine, 4.00; Inositol, 100.00; Ascorbic acid, 50.00-250.00;Sucrose, 10000-30000.00; Kinetin, 0.02-4.0; Benzyl amino purine,0.5-5.0; Agar, 7000.00-12000, all added in weight as milligrams and thefinal volume made upto one litre by adding water.

Yet another embodiment of the present invention wherein the pH of thesaid compositions is in the range of 5.4 to 6.2.

Still another embodiment of the present invention, for said compositionswherein at least one plant growth regulator that is employed is selectedfrom the group consisting of auxins and cytokinins, and a combinationthereof.

Yet another embodiment of the present invention, the compositionswherein the growth regulator auxin is selected from the group consistingof indole acetic acid, indole butyric acid and naphthalene acetic acid,at a concentration range varying between 0.01 to 10 mg/L w/v.

Still another embodiment of the present invention, the compositionswherein the growth regulator cytokinins is selected from the groupconsisting of 6-benzylamino-purine, gamma-gamma-dimethyl allylaminopurine, isopentinyl adenine and kinetin, at a concentration rangevarying between 0.01 mg/L up to 20 mg/L.

The present invention also provides a process for producing acomposition for inducing organized shoots for micro-propagation ofscented Pandanus amaryllifolius, said process comprising mixing thefollowing ingredients NH₄NO₃, in the range of 750-2000,KNO_(3, 800)-1800; H₃BO₄, 6.2; KH₂PO₄, 170.0; KI, 0.83; Na₂MoO₄, 0.25;CoCl₂, 0.025; CaCl₂ 2H₂O, 440-800; MgSO₄ 7H₂O, 370.00; MnSO₄ 4H₂O,22.30; MnSO₄ 4 H₂O, 22.30; ZnSO₄, 8.60; CuSO₄ 5H₂O, 0.025; Na₂ EDTA,37.35; FeSO₄ 7H₂O, 27.85; Thiamine HCl, 1.00; Pyridoxine HCl, 1.00;Nicotinic acid, 1.00; Glycine, 4.00; Inositol, 100.00; Ascorbic acid,50.00-250.00; Glutamine, 50-200; Sucrose, 10000-40000; Soluble polyvinylpyrrolidone 500-4000; Benzyl amino purine, 0.01-2.0; GELRITE-™(phytagel), 2000.00 all added in weight as milligrams and the finalvolume made up to one litre by adding glass distilled water.

The present invention further provides a process for producing acomposition for inducing multiple shoots for the micro-propagation ofscented Pandanus amaryllifolius, said process comprising mixing of thefollowing ingredients:NH₄NO₃, in the range of 1000-2500, KNO₃, 500-2000;H₃BO₄, 6.2; KH₂PO₄, 170.0; KI, 0.83; Na₂MoO₄, 0.025; CoCl₂, 0.025; CaCl₂2H₂O, 440.00; MgSO₄ 7H₂O, 370.00; MnSO₄ 4H₂O, 22.30; MnSO₄ 4 H₂O, 22.30;ZnSO₄, 8.60; CuSO₄ 5H₂O, 0.025; Na₂ EDTA, 37.35; FeSO₄ 7H₂O, 27.85;Thiamine HCl, 1.00; Pyridoxine HCl, 1.00; Nicotinic acid, 1.00; Glycine,4.00; Inositol, 100.00; Ascorbic acid, 50.00-250.00; Sucrose,10000-30000.00; Kinetin, 0.02-4.0; Benzyl amino purine, 0.5-5.0; Agar,7000.00-12000, all added in weight as milligrams and the final volumemade upto one litre by adding water.

An embodiment of the present invention, wherein for the above processesthe pH of the medium is in the range of 5.4 to 6.2.

Another embodiment of the present invention, wherein for the aboveprocesses at least one plant growth regulator that is employed in themedium is selected from the group consisting of auxins and cytokinins,and a combination thereof.

Yet another embodiment of the present invention, wherein for the aboveprocesses the growth regulator auxin is selected from the groupconsisting of indole acetic acid, indole butyric acid and naphthaleneacetic acid, at a concentration range varying between 0.01 to 10 mg/Lw/v.

Still another embodiment of the present invention, wherein for the aboveprocesses the growth regulator cytokinins is selected from the groupconsisting of 6-benzylamino-purine, gamma-gamma-dimethyl allylaminopurine, isopentinyl adenine and kinetin, at a concentration rangevarying between 0.01 mg/L up to 20 mg/L.

The following examples are given by way of illustration of the presentinvention and therefore, should not be construed to limit the scope ofthe invention.

EXAMPLE 1

Medium for establishment of aseptic cultures: Shoots from healthyPandanus plants are collected and the 0.5-1.5 cm long shoot axis withlateral/terminal shoot buds are treated to remove bacterial/fungal(contaminants) by dipping the shoot axis in 0.1% w/v of freshly preparedaqueous mercuric chloride solution for 6 minutes followed by severalrinses with sterilised distilled water. The meristems/shoot buds areaseptically removed by cutting with sterile scalpel under laminar flowand cultured on a first nutrient medium, namely Murashige and Skoog'smedium. This first culture medium is prepared by dissolving in water thefollowing nutrients: Concentration in medium Constituents(milligram/Liter) NH₄NO₃ 1650 KNO₃ 1425 H₃BO₄ 6.2 KH₂PO₄ 170.0 KI 0.83Na₂MoO₄ 0.25 CoCl₂ 0.025 CaCl₂2H₂O 440.00 MgSO₄7H₂O 370.00 MnSO₄4H₂O22.30 ZnSO₄7H₂O 8.60 CuSO₄5H₂O 0.025 Na₂ EDTA 37.35 FeSO₄7H₂O 27.85Thiamine HCl 1.00 Pyridoxine HCl 1.00 Nicotinic acid 1.00 Glycine 4.00Inositol 100.00 Sucrose 30000.00 Glutamine 100.0 Benzyl amino purine 0.5Soluble polyvinyl pyrrolidone 1000.00 Gelrite 2000.00

The pH of the medium is adjusted to 5.8 prior to adding gelrite andboiled to homogeneity, dispensed to culture bottles and the screw-cappedculture bottles with medium are autoclaved at 121 degree C., 15 lb/cm²for 20 minutes. The surface sterilized shoot cuttings are then placed,one at a time using sterile forceps, in a sterile petri-dish in alaminar flow, the shoot tip and small shoot-bud explants are separatedfrom the shoot cuttings with the help of sharp scalpel blades, furtherappropriately trimmed to obtain small shoot buds of 0.5-2 millimetresize and placed on sterile medium contained in the culture bottles. Theshoot buds placed on the sterile nutrient medium are incubated in darkat 25±2 degree C. until tiny shoot cultures are established that maytake 4-6 weeks period. In addition to forming tiny shoot buds, there maybe a ten-fold multiplication in the number of shoot buds during thistreatment. The response of initial explants towards the formation ofshoot buds and tiny shoots during this treatment may be up to 10%.

The various other levels and combinations of kinetin and benzyl aminopurine and other growth regulators such as 2,4-dichlorophenoxy aceticacid, indole acetic acid, indole butyric acid, added to the nutrientmedium, may result in poor response or lesser percentage of response toform shoot cultures or abnormal callus growth without forming shoots.

The medium without any growth regulator or lesser sucrose levels may notsupport the establishment of shoot cultures from shoot tip/shoot budexplants.

EXAMPLE 2

Medium for multiplication of aseptic shoots: The shoot culturesestablished as in example 1 has to be used as the explant material forthe clonal multiplication of Pandanus shoots. The shoot buds establishedas in Example 1 are removed aseptically in a laminar flow chamber andplaced on a sterile petri plate. If there is clustered shoot budformation, each bud is separated by cutting using sterile scalpel. Aboutthree to four shoot buds are then placed on a liquid nutrient mediumcontaining the macroelements, microelements, vitamins and sucrose ofMurashige and Skoog medium (MS, 1962, Physiol. Plantarum 15:473-497) aresimilar to those in Example 1 with the following nutrients in the secondmedium is prepared by dissolving in water the following nutrients:Concentration in medium Constituents (milligram/Liter) NH₄NO₃ 1650.00KNO₃ 1425.00 H₃BO₄ 6.2 KH₂PO₄ 170.0 KI 0.83 Na₂MoO₄ 0.25 CoCl₂ 0.025CaCl₂2H₂O 440.00 MgSO₄7H₂O 370.00 MnSO₄4H₂O 22.30 ZnSO₄7H₂O 8.60CuSO₄5H₂O 0.025 Na₂ EDTA 37.35 FeSO₄7H₂O 27.85 Thiamine HCl 1.00Pyridoxine HCl 1.00 Nicotinic acid 1.00 Glycine 4.00 Inositol 100.00Sucrose 20000.00 Kinetin 1.0 Benzyl amino purine 1.5 Agar-agar 8000.00

The pH of the second medium is adjusted to 5.8 and boiled tohomogeneity, dispensed about 40 millilitre to conical flasks and themedium autoclaved at 121 degree C., 15 lb/cm² for 20 minutes.

The shoots are incubated in the second nutrient medium and incubatedunder a photoperiod of 16 h with an illumination of 2000 lux at 25±2degree C. for a period of 30-45 days by which time each shoot formsnearly ten additional shoots. The shoots are then separated aseptically,the larger ones are transferred to a third nutrient medium for shootelongation as well as rooting as in Example 3, and the smaller ones aretransferred to the second medium and grown for a period of 3-6 weeks,the process is repeated to obtain a large number of shoots.

EXAMPLE 3

Medium for root induction and partial hardening: The larger shoots of2-3 cm length multiplied as in Example 2 are transferred to a thirdmedium developed for rooting and partial hardening containing thefollowing nutrients in water: Stock solution Milliliter volume of stockStock Constituents (grams/liter) solution in 1 liter medium A NH₄NO₃82.5 10 B KNO₃ 95.0 10 C H₃BO₄ 1.24 2.5 KH₂PO₄ 24.0 KI 0.166 Na₂MoO₄0.55 CoCl₂ 0.005 D CaCl₂2H₂O 88.0 2.5 E MgSO₄7H₂O 74.0 2.5 MnSO₄4H₂O4.46 ZnSO₄ 1.72 CuSO₄5H₂O 0.005 F Na₂ EDIA 7.4 2.5 FeSO₄7H₂O 5.57 GThiamine HCl 0.2 2.5 Pyridoxine HCl 0.2 Nicotinic acid 0.2 Glycine 0.8Sucrose 10 g/L Agar 10 g/L

The plantlets were nurtured for 3 weeks at 25-30° C. under 2000 luxillumination where 100% rooting may be encountered which is accompaniedby further growth of shoots. The rooted shoots are removed from thecontainer, washed in running tap water, and then transferred to soilcompost containing 1 part of red soil and 2 parts of garden humus, andkept in a place with 90% humidity for another three weeks period. Thehardened plantlets may be transferred to green house till they areplanted in the field.

The Main Advantages of the Invention

1. The present invention provides, for the first time, a mediumformulation that efficiently supports shoot bud establishment to formaseptic shoot cultures of Pandanus amaryllifolius.

2. The present invention also provides an efficient process for shootmultiplication allowing 10-fold increase in the shoot number in 4 weeksperiod, which is a high rate of multiplication using tissue culturetechnology.

3. The process of the present invention involves a nutrient mediumformulation for continuous multiplication of Pandanus plants throughoutthe year.

4. The process of the present invention allows producing Pandanusamaryllifolius planting material free from saprophytic, fungal andbacterial diseases.

1-8. (canceled)
 9. A composition for inducing organized shoots formicro-propagation of scented Pandanus amaryllifolius, said compositioncomprising: NH₄NO₃, in the range of 750-2000, KNO₃, 800-1800; H₃BO₄,6.2; KH₂PO₄, 170.0; KI, 0.83; Na₂MoO₄, 0.25; CoCl₂, 0.025; CaCl₂ 2H₂O,440-800; MgSO₄ 7H₂O, 370.00; MnSO₄ 4H₂O, 22.30; MnSO₄ 4 H₂O, 22.30;ZnSO₄, 8.60; CuSO₄ 5H₂O, 0.025; Na₂ EDTA, 37.35; FeSO₄ 7H₂O, 27.85;Thiamine HCl, 1.00; Pyridoxine HCl, 1.00; Nicotinic acid, 1.00; Glycine,4.00; Inositol, 100.00; Ascorbic acid, 50.00-250.00; Glutamine, 50-200;Sucrose, 10000-40000; Soluble polyvinyl pyrrolidone 500-4000; Benzylamino purine, 0.01-2.0; GELRITE™ (phytagel), 2000.00 all added in weightas milligrams and the final volume made up to one litre by adding glassdistilled water.
 10. A composition for inducing multiple shoots for themicro-propagation of scented Pandanus amaryllifolius, said compositioncomprising: NH₄NO₃, in the range of 1000-2500, KNO₃, 500-2000; H₃BO₄,6.2; KH₂PO₄, 170.0; KI, 0.83; Na₂MoO₄, 0.25; CoCl₂, 0.025; CaCl₂ 2H₂O,440.00; MgSO₄ 7H₂O, 370.00; MnSO₄ 4H₂O, 22.30; MnSO₄ 4 H₂O, 22.30;ZnSO₄, 8.60; CuSO₄ 5H₂O, 0.025; Na₂ EDTA, 37.35; FeSO₄ 7H₂O, 27.85;Thiamine HCl, 1.00; Pyridoxine HCl, 1.00; Nicotinic acid, 1.00; Glycine,4.00; Inositol, 100.00; Ascorbic acid, 50.00-250.00; Sucrose,10000-30000.00; Kinetin, 0.02-4.0; Benzyl amino purine, 0.5-5.0; Agar,7000.00-12000, all added in weight as milligrams and the final volumemade up to one litre by adding water.
 11. The composition of claims 9and 10, wherein the pH of the composition is in the range of 5.4 to 6.2.12. The composition of claims 9 and 10, wherein at least one plantgrowth regulator that is employed in the medium is selected from thegroup consisting of auxins and cytokinins, and a combination thereof.13. The composition of claims 9 and 10, wherein the growth regulatorauxin is selected from the group consisting of indole acetic acid,indole butyric acid and naphthalene acetic acid, at a concentrationrange varying between 0.01 to 10 mg/L w/v.
 14. The composition of claims9 and 10, wherein the growth regulator cytokinins is selected from thegroup consisting of 6-benzylamino-purine, gamma-gamma-dimethylallylamino purine, isopentinyl adenine and kinetin, at a concentrationrange varying between 0.01 mg/L up to 20 mg/L.
 15. A process forproducing a composition for inducing organized shoots formicro-propagation of scented Pandanus amaryllifolius, said processcomprising mixing the following ingredients NH₄NO₃, in the range of750-2000, KNO₃, 800-1800; H₃BO₄, 6.2; KH₂PO₄, 170.0; KI, 0.83; Na₂MoO₄,0.25; CoCl₂, 0.025; CaCl₂ 2H₂O, 440-800; MgSO₄ 7H₂O, 370.00; MnSO₄ 4H₂O,22.30; MnSO₄ 4 H₂O, 22.30; ZnSO₄, 8.60; CUSO₄ 5H₂O, 0.025; Na₂ EDTA,37.35; FeSO₄ 7H₂O, 27.85; Thiamine HCl, 1.00; Pyridoxine HCl, 1.00;Nicotinic acid, 1.00; Glycine, 4.00; Inositol, 100.00; Ascorbic acid,50.00-250.00; Glutamine, 50-200; Sucrose, 10000-40000; Soluble polyvinylpyrrolidone 500-4000; Benzyl amino purine, 0.01 -2.0; GELRITE™(phytagel), 2000.00 all added in weight as milligrams and the finalvolume made up to one litre by adding glass distilled water.
 16. Aprocess for producing a composition for inducing multiple shoots for themicro-propagation of scented Pandanus amaryllifolius, said processcomprising mixing of the following ingredients: NH₄NO₃, in the range of1000-2500, KNO₃, 500-2000; H₃BO₄, 6.2; KH₂PO₄, 170.0; KI, 0.83; Na₂MoO₄,0.25; CoCl₂, 0.025; CaCl₂ 2H₂O, 440.00; MgSO₄ 7H₂O, 370.00; MnSO₄ 4H₂O,22.30; MnSO₄ 4 H₂O, 22.30; ZnSO₄, 8.60; CuSO₄ 5H₂O, 0.025; Na₂ EDTA,37.35; FeSO₄ 7H₂O, 27.85; Thiamine HCl, 1.00; Pyridoxine HCl, 1.00;Nicotinic acid, 1.00; Glycine, 4.00; Inositol, 100.00; Ascorbic acid,50.00-250.00; Sucrose, 10000-30000.00; Kinetin, 0.02-4.0; Benzyl aminopurine, 0.5-5.0; Agar, 7000.00-12000, all added in weight as milligramsand the final volume made up to one litre by adding water.
 17. Theprocess of claims 15 and 16, wherein the pH of the medium is in therange of 5.4 to 6.2.
 18. The process of claims 15 and 16, wherein atleast one plant growth regulator that is employed in the medium isselected from the group consisting of auxins and cytokinins, and acombination thereof.
 19. The process of claims 15 and 16, wherein thegrowth regulator auxin is selected from the group consisting of indoleacetic acid, indole butyric acid and naphthalene acetic acid, at aconcentration range varying between 0.01 to 10 mg/L w/v.
 20. The processof claims 15 and 16, wherein the growth regulator cytokinins is selectedfrom the group consisting of 6-benzylamino-purine, gamma-gamma-dimethylallylamino purine, isopentinyl adenine and kinetin, at a concentrationrange varying between 0.01 mg/L up to 20 mg/L.